用于研究尿嘧啶-DNA糖基化酶的修饰底物探针

Modified substrates as probes for studying uracil-DNA glycosylase.

作者信息

Kubareva E A, Volkov E M, Vinogradova N L, Kanevsky I A, Oretskaya T S, Kuznetsova S A, Brevnov M G, Gromova E S, Nevinsky G A, Shabarova Z A

机构信息

A.N. Belozersky Institute of Physical and Chemical Biology, Moscow State University, Russia.

出版信息

Gene. 1995 May 19;157(1-2):167-71. doi: 10.1016/0378-1119(94)00771-j.

DOI:10.1016/0378-1119(94)00771-j

PMID:7607485

Abstract

In order to study the mechanism of action of uracil-DNA glycosylase (UDG) from human placenta, single-stranded (ss) and double-stranded (ds) oligodeoxyribonucleotides (oligos), containing deoxyuridine (dU) and a wide variety of their analogs were used. It was shown that UDG has a twofold preference for ss oligos over ds oligos and a twofold preference for intermolecular duplexes over similar hairpin-like duplexes. The replacement of dU with 1-(beta-D-2'-deoxy-threo-pentofuranosil)uracil (xU) or 1-(beta-D-3'-deoxy-threo-pentofuranosil)uracil (tU), which results in a change in sugar hydroxyl configuration, has no influence on UDG binding to such substrates, but inhibits uracil removal. A oligo containing 2'-deoxy-2'-fluorouridine (flU), with a 3'-endo conformation of modified sugar is recognized by UDG 100-200-fold less efficiently than the natural ones. F or Br atoms or a methyl group were introduced at position 5 of a dU residue in an oligo. It was shown that the nature of a substituent at this position is essential for UDG function.

摘要

为了研究人胎盘尿嘧啶-DNA糖基化酶（UDG）的作用机制，使用了含有脱氧尿苷（dU）及其多种类似物的单链（ss）和双链（ds）寡脱氧核糖核苷酸（oligos）。结果表明，UDG对单链寡核苷酸的偏好是双链寡核苷酸的两倍，对分子间双链体的偏好是类似发夹状双链体的两倍。用1-(β-D-2'-脱氧-苏式-戊呋喃糖基)尿嘧啶（xU）或1-(β-D-3'-脱氧-苏式-戊呋喃糖基)尿嘧啶（tU）取代dU，这会导致糖羟基构型发生变化，对UDG与此类底物的结合没有影响，但会抑制尿嘧啶的去除。含有2'-脱氧-2'-氟尿苷（flU）的寡核苷酸，其修饰糖具有3'-内型构象，被UDG识别的效率比天然寡核苷酸低100-200倍。在寡核苷酸的dU残基的5位引入F或Br原子或甲基。结果表明，该位置取代基的性质对UDG功能至关重要。